Method and apparatus for sorting and analyzing particles in an aerosol with redundant particle analysis

ABSTRACT

A method and apparatus for sorting and performing redundant analysis of particles in an aerosol is disclosed. Redundant analysis reduces the possibility of false positive analyses, which is advantageous in the art. The apparatus may comprise an aerosol concentrator, an optical particle analyzer, an electrosprayer and a charged particle analyzer. A method according to the invention may comprise delivering a concentrated aerosol stream to an optical particle analyzer; analyzing each particle of interest and selectively triggering an electrosprayer to electrospray each particle of interest; adding a charge to the particle, which is then moved by electrostatic forces to a charged particle analyzer; and performing a second, redundant analysis of each charged particle collected on the charged particle analyzer to confirm the identity of the particle of interest. The apparatus and method may also be adapted to perform redundant analysis of disguised particles that are coated to disguise their payload.

FIELD OF THE INVENTION

The field of the invention is methods and apparatus for sorting andanalyzing particles in an aerosol. An aerosol is generally defined asany gaseous fluid containing particles. For purposes of the presentinvention, most samples of ambient air are aerosols. A prompt analysisof particles in an aerosol has many applications. Terrorists and bombersmay seek to plant explosive devices so as to cause destruction, injuryand death. If such terrorist or bomber has particles of the explosive onhis or her person, then an analysis of the particles in the airsurrounding the person may allow for the early detection of a terroristor bomber. If the terrorist seeks to cause injury of a targeted group byexposure to a chemical agent such as nerve gas, a pathogen such as avirus or bacteria, or a radioactive composition, then prompt detectionof the attack may allow defensive measures to be taken, so as tominimize the injury and death.

BACKGROUND OF THE INVENTION

This invention pertains to the field of collecting, sorting andanalyzing particles in an aerosol and, more particularly, to a methodand apparatus for collecting an aerosol, sorting particles within theaerosol and subjecting the particles to redundant analysis for thepurpose of identifying the particles.

The separation and capture of particles or aerosols from air or otherfluid streams are of concern in two contexts: first, in determining thetype and concentration of such particles/aerosols and, second, incleansing the fluid stream for subsequent use. For example, thedetection or extraction of airborne biological or chemical warfareagents, biological contamination in confined spaces, such as aircraft orhospitals, or industrial pollutants (either in ambient air or insmokestacks) may be required in various scenarios.

Much effort has been expended in the past in the collection of aerosols,the extraction of particles from aerosols and the analysis andidentification of particles in aerosols.

The prior art includes methods and apparatus for analyzing particles ingases in possible target areas, including public buildings, criticalinfrastructure such as water supply facilities, and defensiveinstallations such as military bases. The prior art includes sampling ofthe atmosphere from possible target areas. In some prior arttechnologies, all of the particles from a sample are isolated, and thenthe particles are analyzed. In other prior art methods, the volume ofthe sample is reduced, and a representative portion of the particles isanalyzed.

The prior art recognizes that the avoidance of false positives is ofcritical importance. A false positive with respect to explosiveparticles, pathogen particles, radioactive particles, and any otheradverse particles, may lead to unnecessary evacuations and possibleinjury and death. In addition, if a particular method and apparatusresult in repeated false positives, then the level of confidence in thatmethod and apparatus may be degraded to the point that vigorousresponses to protect the public will become difficult to achieve.However, to date, none of the prior art adequately addresses the problemof false positives.

SUMMARY OF THE INVENTION

A method and apparatus for sorting and analyzing particles in an aerosolwith redundant particle analysis is achieved by the present invention.This redundant particle analysis reduces false positives.

In one embodiment of the invention, a stream of an aerosol enters thefirst concentrator of the apparatus. The first concentrator removes themajority of the gas from the aerosol to provide a first concentratedaerosol that is fed to a first optical analyzer. In another embodimentof the invention, a stream of an aerosol passes through multipleconcentrators to provide a first concentrated aerosol.

The stream of the first concentrated aerosol passes through the firstoptical analyzer of the apparatus, which exposes the particles in thefirst concentrated aerosol to one or more lasers. The lasers maycomprise infrared lasers, ultraviolet lasers, and combinations thereofas well as other lasers. The first optical analyzer analyzes thescattering and/or absorption of one or more of the laser beams that iscaused by the particles, and recognizes any particles of interest basedon scattering and/or absorption characteristics.

The first concentrated aerosol flows from the first optical analyzer tothe first electrosprayer, which comprises a first electrospray nozzle, asupply of a first electrospray fluid, and a first electrospray trigger.In a preferred embodiment, the electrosprayer also comprises acontroller and a high voltage power supply. In one embodiment of theinvention, the first electrosprayer may comprise multiple electrosprayfluid reservoirs, with different electrospray fluids suited foradministering to different types of particles of interest. The firstelectrospray trigger fires the first electrospray nozzle to apply thefirst electrospary fluid to one or more selected particles of interest.The timing of the firing may be calculated based upon the distancebetween the laser scattering in the first optical analyzer and the firstelectrosprayer, as well as the flow rate of the first concentratedaerosol through the apparatus. The application of an electrospray fluidto a particle of interest causes the particle to become electricallycharged and attracted by electrostatic forces to a first chargedparticle analyzer located downstream of the electrosprayer. The firstcharged particle analyzer is preferably a micropillar array. Micropillararrays are disclosed in U.S. Pat. No. 6,110,247.

The method and apparatus of the present invention are particularlyadvantageous in allowing for redundant particle analysis. A particle ofinterest is initially detected and analyzed by the first opticalanalyzer. This analysis is later confirmed when the same particle iscoated with an electrospray fluid and attracted to the first chargedparticle analyzer, on which a second analysis of the same particle maybe performed. The redundant analysis of a particle of interest by boththe first optical particle detector and the first charged particleanalyzer significantly reduces the possibility of a false positive andimproves over the prior art.

The initial analysis by the first optical analyzer may be based upon acomparison of the scattering and/or absorption caused by the particleunder interrogation to the known results of interrogation of particlesof interest. Particles of interest include adverse particles such asparticles of explosives, biological particles such as viruses andbacteria, and radioactive particles, each of which may be known to causeinjury, disease and/or death.

In addition, the present invention is capable of dealing with particlesof interest that may be disguised particles. Terrorists and the likeseeking to cause disease, injury or death, may disguise adverseparticles with coatings that are designed to avoid detection. Forexample, an adverse particle known to cause disease, injury or death,may be coated with a time-release substance so as to avoid detection ofthe adverse particle. The first optical analyzer may be programmed torecognize such coatings, and designate the relevant particle to be aparticle of interest for further analysis, such as when the particle ofinterest is collected on the first charged particle analyzer.

In one embodiment of the invention, the first optical analyzer maycomprise an ultraviolet laser, an infrared laser, and a source ofvisible electromagnetic radiation to allow interrogation of theinfrared, ultraviolet and visible spectra generated by the particles inthe first concentrated aerosol stream.

In another embodiment of the invention, the first optical analyzer maycomprise a split beam. For example, the beam from an infrared laser maybe split into a first beam and a second beam, so that the firstconcentrated aerosol stream passes first through the first beam, andsubsequently through the second beam. The distance between the firstbeam and the second beam may be fixed. The first optical analyzer maythen compare the interrogation of particles by the first beam and thesecond beam. The particles in the first concentrated aerosol stream mayhave unique spectra (both absorption and transmission). Thus, theprecise time at which a particular particle passes through the firstbeam may be compared to the precise time at which that particle passesthrough the second beam, so as to allow precise calculation of the speedof the particle as it enters the first electrosprayer. This may allowfor a more precise triggering of the electrospray nozzle, and thussignificantly reduces the possibility that a particle not considered tobe of interest is coated with an electrospray fluid.

In one embodiment of the invention, the first charged particle analyzermay comprise multiple rows of micropillars. The diameters of themicropillars may decrease in each row. Thus, the diameter of the micropillars in the first row may be larger than the diameter in the secondrow, and the diameter of the micropillars in the second row may belarger than the diameter of the pillars in the third row, and so forth.This may facilitate the collection of larger particles on the first row,particles of smaller size on the second row, particles of still smallersize on the third row, and so forth.

The particles on the micropillars may undergo a second analysis in anumber of different manners. The electrospray fluid may comprise afluorescent tag that fluoresces when it becomes attached to a particleof a particular composition. For example, there may be a source ofelectrospray fluid that comprises a fluorescent tag that fluoresces whenit comes into contact with a particle of a particular pathogen. If thisfluorescence is detected on the micropillars, then the presence of thatpathogen has been detected and/or confirmed.

The particles on the micropillars may also be subjected to Ramanspectroscopy. Generally speaking, Raman spectroscopy looks for a shiftin the spectra caused by a particular particle and may use radiation inthe ultraviolet, infrared or visible range of the electromagneticspectrum.

The redundant particle analysis of the method and apparatus of thepresent invention may require an additional step of lysis when adisguised particle is detected. A disguised particle may comprise anadverse particle such as a bacterium, which may be referred to as thepayload, and a coating, which may be referred to as the disguise. If adisguised particle is detected by the first optical analyzer, then thefirst electrosprayer may be triggered to coat the disguised particlewith an electrospray fluid, which causes the disguised particle todevelop an electrical charge and be attracted to the first chargedparticle analyzer by electrostatic forces. The payload within adisguised particle may be impossible to verify unless the disguise isremoved by removing the coating of the particle.

The disguised particle may be exposed to plasma lysing, which breachesthe coating on the particle and exposes the payload. The payloadparticle may then be further analyzed. For example, the payload particlemay be introduced into a second concentrated aerosol stream, which isfed into a second optical analyzer, then into a second electrosprayer,and subsequently into a second charged particle analyzer. This may allowfor the sorting and analysis of the payload particle in the same mannerthat particles are sorted and analyzed by the first optical analyzer,the first electrosprayer, and the first charged particle analyzer.

In another embodiment of the present invention, the payload particle maybe extracted from the first charged particle analyzer into a liquid, andanalyzed by polymerase chain reaction (PCR), antigen assay, and/or otherbiosensor techniques. Other embodiments of the invention may comprisemultiple optical analyzers, multiple electrosprayers and multiplecharged particle analyzers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b are flow diagrams of different embodiments of thepresent invention.

FIG. 2 is a schematic view of the present invention.

FIG. 3 illustrates an optical particle analyzer used in the presentinvention.

FIG. 4 is a graph showing data collected from scattering a split-beamlaser with particles in an aerosol.

FIG. 5 illustrates an electrosprayer and a charged particle analyzerused in the present invention.

FIG. 6 is an IR spectroscopy graph comparing the data collected from acontrol spore group to data collected from spores of Bacillus globigii.

DETAILED DESCRIPTION

As used in this specification, the following terms shall have thefollowing definitions unless the context indicates otherwise:

Aerosol—a suspension of particles in a fluid;

Particle—any separately identifiable solid, liquid, aerosol or othercomponent entrained in a fluid stream that has a greater mass than thefluid forming the fluid stream, and is the subject of separation andcollection for analysis;

Fluid—any fluid susceptible to fluid flow, which may comprise acombination of liquids and gases, and which may entrain foreignparticles therein.

The first embodiment of the present invention comprises an apparatus forsorting and analyzing particles in an aerosol. The four main componentsof the apparatus generally include a concentrator, an optical particleanalyzer, an electrosprayer, and a charged particle analyzer.

As shown in FIG. 1 a, the first concentrator 200 collects ambientaerosol sample 100 and provides concentrated aerosol to the firstoptical analyzer 300, which is located downstream from the aerosolconcentrator 200. An optional additional concentrator 250 may also bepresent between the first concentrator and first optical analyzer inorder to further concentrate the aerosol 100. Particles in theconcentrated aerosol passing through the optical particle analyzer 300are then passed through the electrosprayer 400, which is locateddownstream from the optical particle analyzer 300. Upon passing throughthe electrosprayer 400, selected particles in the concentrated aerosolare sprayed with an electrospray fluid, which preferably adds a chargeto the selected particles. Next, the electrosprayed particles arecollected on the first charged particle analyzer 500 located downstreamfrom the electrosprayer 400. The first charged particle analyzer 500provides the opportunity to perform a second analysis on the chargedparticles collected on the first charged particle analyzer 500, therebyconfirming earlier analysis performed by the first optical analyzer 300.

As also shown in FIG. 1, analysis of the particles that are collected onthe first charged particle analyzer may further comprise bio-analysis600, such a polymerase chain reaction (PCR), antigen assay or otherknown bio-analysis techniques. Particles that are not disguised by acoating are suitable for bio-analysis on the first charged particleanalyzer.

However, in the case where the particle is disguised by, for example, acoating, the disguise is breached to expose the payload as shown in FIG.1 b. Breaching 650 the coating of the particle may be accomplished by,e.g., plasma lysis, which will penetrate the coating and expose thepayload particle. The payload particle may be passed to a secondconcentrator 700, second optical analyzer 800, second electrosprayer 900and second charged particle analyzer 1000. The payload particle is thusanalyzed and identified in the same manner as described above withrespect to a non-disguised particle. As with the first concentrator, thefirst optical analyzer, the first electrosprayer and the first chargedparticle analyzer, the present invention allows the second concentrator,second optical analyzer, second electrosprayer and second chargedparticle analyzer to perform redundant analysis of the payload particle.

FIG. 2 illustrates a schematic of components of the apparatus accordingto the present invention. The first concentrator 200 collects an ambientaerosol, concentrates the aerosol by exhausting fluid withoutsignificant loss of particles and then passes the concentrated aerosolto the first optical analyzer 300. In FIG. 2, the aerosol passed intothe system from the concentrator comprises particles of two types, onebeing of interest and the other not. The optical particle analyzercomprises at least one laser 301 and a photomultiplier tube (PMT) orother suitable optical or electronic detector 302. The laser 301 firesone or more laser beams at the passing particles. The detector 302 thenmeasures the scattering and/or absorption of each laser to determine theidentity of each particle. Along with scattering and absorptioncharacteristics, size and velocity of the particles as well asfluorescent signatures of the particles may also be used to identify anyparticle of interest.

Having conducted the first analysis of each particle, the first opticalanalyzer 300 sends a signal to the electrosprayer 400 via datatransferring line 303, and the signal is used to instruct when and atwhich particles to fire the electrospray fluid. These parameters aredetermined primarily by, e.g., the velocity measurements of theparticles measured/calculated by the first optical analyzer 300. Asshown in FIG. 2, the data collected from the first optical analyzer 300has instructed the electrosprayer to fire electrospray fluid atparticles of interest 101, but not other particles 102.

Electrospray fluid is preferably stored in a reservoir 402 and suppliedto the electrospray nozzle for discharging at particles. Thenon-electrosprayed particles 102 continue downstream in the apparatus.The electrosprayed particles 101 are electrically charged and attractedto the first charged particle analyzer 500. The other particles 102 arecollected in a separate region, or may be discharged from the apparatus.

Turning now to a more detailed description of each component of anapparatus according to the present invention, the concentrator is acomponent of the apparatus for extracting aerosol from the surroundingenvironment and exhausting predominantly fluid from the aerosol. Byexhausting predominantly fluid without also exhausting the particles,the concentrator produces a concentrated aerosol with only a fraction ofthe fluid found in the ambient aerosol. For example, the concentratormay exhaust 90% of the fluid component of the ambient aerosol, resultingin a concentrated aerosol having the same number of particles found inthe ambient aerosol but with only 10% of fluid of the ambient aerosol.

The concentrator may be designed such that the fluid exhausted from theconcentrator does not contain particles smaller than a predeterminedsize. That is to say, the concentrator may be selected such that few orno particles smaller than a specified diameter are lost in the exhaustflow and most or all particles below the specified diameter are retainedin the concentrated aerosol that is passed to the optical analyzer. Inthis manner, the concentrator may serve as a first sorter for sortingparticles in the aerosol and may be selected according to the needs ofthe situation.

In a preferred embodiment, the concentrator is a virtual impactor suchas disclosed by U.S. Pat. No. 7,178,380. Conventional impactors comprisea nozzle above an impaction plate. In a virtual impactor, the impactionplate is replaced with a receiving nozzle that has a virtual impactionsurface that is the inlet of a receiving tube. Even more preferably, theconcentrator is a radial axisymmetric virtual impactor.

The present invention further comprises an optical analyzer, which islocated downstream of the concentrator. As shown in FIG. 3, the opticalanalyzer 300 generally comprises a laser 310, a PMT 304 and an area 306between the laser 310 and the PMT 304 that allows for particles to passby and be struck by the laser beam emanating from laser 310. The laser310 may be any laser suitable for use in elastic scattering, forexample, a Nd-YAG laser, an infrared wavelength laser, or quantumcascade lasers at infrared frequencies. The laser 310 may also beprotected from the aerosol by barrier flows of air, or air curtains.These air curtains prevent particles in the concentrated aerosol fromcontacting or damaging the laser.

In the embodiment shown, the laser 310 produces rapid pulses or acontinuous beam of a single beam laser, which is split by beamsplitters308, although the laser need not be split. The rapid pulses orcontinuous beam of the split beam laser collide with the passingparticles, causing scattering of light. The collision of the beam withthe particles may also lead to absorption of the beam by the particle.The PMT 304 measures the light scattering and/or absorption and canperform a first identification of the particles based upon thesemeasurements.

FIG. 4 shows measurements taken from light scattering in the opticalanalyzer, wherein the distance between pulses correlates with velocityand the peak height correlates with particle diameter. Thus, thephysical characteristics of the particles may be measured in the opticalanalyzer.

Also, the inelastic scattering of light may be used to determine thefluorescent signatures of the particles. Certain wavelengths of light inthe laser beam may interact with the particle to cause amino acidslocated on the particle surface, such as tryptophan, to fluoresce, thusindicating the presence of certain amino acids on the particle. Thestimulated fluorescent emission may be characteristic of certainbiomaterials and chemicals of interest, and therefore may be used foridentification and classification purposes. The PMT 304 is capable ofmeasuring the stimulated fluorescent emissions as well as elastic lightscattering and absorption.

As alluded to above, infrared lasers that produce light that ischaracteristically absorbed by certain biomaterials and chemicals ofinterest may also be used alternative to or in combination withfluorescence to further aid in the first analysis of the particles.Again, the PMT 304 is capable of measuring whether infrared light hasbeen absorbed.

Having now performed a first analysis of the particles as well asgathering measurements, e.g., size and velocity, of the particles, thisinformation is used to determine when and at which particles to fire theelectrospray fluid as well as what type of electrospray fluid to fire.The data from the first analysis is transmitted to the electrosprayer,where it is received and processed to select the characteristics of thespray as well as the electrospary fluid to be sprayed. Thus determined,the electrospray nozzle is triggered in order to coat the selectedparticle with the selected electrospray fluid.

Specifically, the velocity measurement is used to determine when to firethe electrospray nozzle based on knowing both the speed at which theparticle of interest is traveling and the distance between theelectrospray nozzle and the point of analysis of the particle into theoptical analyzer. This distance may vary, but is preferably less than 1cm to eliminate diverging flow.

The measurement taken to determine the size of the particle may be usedto ensure that the electrospray fluid is not fired at particles whichare clearly not particles of interest, such as large particles of pollenentrained in the concentrated aerosol.

Other measurements, such as the fluorescent signature measurement, areused to select at which particles to fire the electrospray fluid andalso the type of electrospray fluid to be fired at the particles. Forexample, the fluorescence of a particle upon being subjected to thesplit laser beam indicates the presence of certain components on theparticle which in turn may be used to identify or classify the particle.The fluorescent signature measurement may therefore be used to determinewhether to fire the electrospray fluid at a specific particle andadditionally the type of electrospray fluid to be fired at the particle.

Additionally, as mentioned above, a particle's absorption of infraredlight may be used in a similar manner. Biomaterials and particles ofinterest will absorb wavelengths of infrared light and the detection ofthis absorption may be used to identify or classify the particles. Onceidentified or classified, the information is used to decide whether tofire electrospray fluid at a particle and if so, which electrosprayfluid to fire at a particle.

Following the optical analyzer, the next main component of the apparatusaccording to a preferred embodiment of the invention, is theelectrosprayer which receives the particles after they have traversedthe laser beams in the optical analyzer. The electrosprayer preferablycomprises a controller, a high voltage power supply, at least oneelectrospray nozzle and an electrospray fluid reservoir. Eachelectrospray nozzle is preferably comprised of at least one passage wayfor emitting at least one electrospray fluid.

FIG. 5 illustrates an electrosprayer 410 coupled with a first chargedparticle analyzer 500. The particles move downward in the direction ofthe arrow shown in FIG. 5 and pass through the electrosprayer. Based onthe information collected and processed from the optical analyzer anddiscussed in more detail previously (i.e., particle velocity, particlesize and particle fluorescent signature), an electrospray trigger firesan electrospary nozzle when a selected particle passes by theelectrospray nozzle. The electrospray nozzle fires a short, pulsed burstof electrospray fluid to preferably coat only the specific particlepassing by the electrosprayer at the time of triggering. Preferably, theinitiation and termination of the burst of electrospray fluid occurs inless than one millisecond to ensure selective coating of only theparticle of interest. High frequency operation of the electrosprayerenables higher particle concentration samples to be efficiently sortedand identified.

The electrospray fluid is fired at selected particles in order to add acharge to the selected particles. The electrospray fluid may alsocomprise an immunoassay to aid in the second identification of theparticle. When the electrospray fluid attaches to the particle orparticles, the particles are preferably sufficiently wetted with theelectrospray fluid to prevent denaturing or dehydration resulting indrying out of the labile biomaterial or an immunoassay, if present.Similarly, if a nanoassay such as a polymer that changes the lightscattering of an explosive particle is present on the particle,sufficient wetting will prevent the dehydration of the nanoassay.

Electrospray fluids comprise reagents whose fluorescence, infrared orvisible and other electromagnetic radiation relates to at lease onespecific binding, absorption, or reaction event. Any electrospray fluidcapable of adding a charge to a particle may be used, generallyspeaking.

The electrosprayer may also be capable of firing multiple types ofelectrospray fluid in order to coat different particles with differenttypes of electrospray fluid. In this aspect of the invention, theinformation collected and processed by the optical analyzer istransmitted to the electrosprayer such that the electrosprayer isinstructed to fire a first electrospray at selected particles and tofire a second, different electrospray at other selected particles. Inthis manner, the apparatus of the first embodiment is capable ofperforming a second, redundant identification of multiple particles inthe concentrated aerosol at once. Alternatively, the electrosprayer canbe instructed to fire a first and a second electrospray at the sameparticle, in order to provide multiple means by which to performredundant analysis of the particle. For example, the particle could besprayed with both an immunoassay and an a FRET (fluorescence resonanceenergy transfer) probe, such that subsequent analysis and identificationof the particle can be accomplished by using either of the two coatingsor can be used to confirm the conclusion drawn from the first coating.

In addition, the electrosprayer may also be used to add a nanoassay tocertain particles. The first analysis of the particle can also be usedto initiate the addition of a nanoassay, such as antibodies withfluorescent probes, to the particle to further effect detection duringsubsequent particle analysis. The identification of the particleinteraction with an optional nanoassay can be accomplished optically orelectrically.

In another embodiment, an additional electrosprayer may be placeddownstream of the concentrator and upstream of the optical analyzer. Thepurpose of the additional electrosprayer is to pretreat the particles inthe concentrated aerosol with a fluid capable of selectively interactingwith particles to produce a signature such as fluorescence.

Downstream of the first electrosprayer, the particles are sorted by afirst charged particle analyzer 500 as show in FIG. 5. As shown in theFIG. 5, the first charged particle analyzer 500 is preferably locatedperpendicular to the general direction the particles are traveling asshown by the arrow in FIG. 5. In this configuration, theelectrospray-charged particles must turn 90 degrees from the generaldirection of travel in order to be collected by the charged particleanalyzer 500, which is preferably grounded. This movement of theparticles is achieved due to the electrostatic attraction between thecharged particle analyzer 500 and the electrospray-charged particles.The non-electrosprayed particles not having a charge continue in thedirection of the arrow shown in FIG. 5 and preferably collect on aparticle collector for uncharged particles 502.

The first charged particle analyzer, as well as the particle collectorfor uncharged particles may each be any size suitable for use in theapparatus of the present invention, but the first charged particleanalyzer normally has an area smaller than the area of the particlecollector for uncharged particles. For example, the first chargedparticle analyzer may have an area of about 1.96 mm², while the particlecollector for uncharged particles may have an area of 19.9 mm².

In one aspect of the first embodiment, the first charged particleanalyzer is a micropillar array. Micropillar arrays are disclosed byU.S. Pat. No. 6,110,247. The micropillars may be cleaned in between usesby using an ionized gas.

With the selected particles now sorted and collected on the firstcharged particle analyzer, the selected particles may be subjected toredundant analysis for the purpose of identifying and/or confirming theselected particles. In a preferred embodiment, any suitable means ofbio-analysis, such as PCR, antigen assay, and/or other biosensortechniques may be used.

If a particle of interest is disguised, i.e., comprises a coating thatprevents analysis of the payload, then the apparatus of the inventionmay perform plasma lysing of the coating on the disguised particle toexpose the payload. The plasma lysing breaches the coating of theparticle and exposes any interstitial contents for analysis. As anexample, a bacterial spore can be lysed by an ionized gas to release thechromosomal nucleic acid and core proteins. Plasma lysis of particles isdisclosed by U.S. Pat. No. 5,989,824.

The present invention provides for redundant analysis of the payload ofdisguised particles, and may include a second concentrator, secondoptical analyzer, second electrosprayer and second charged particleanalyzer as shown in FIG. 1 b and discussed above. Furthermore,duplicate samples may be processed and preserved for polymerase chainreaction replication and identification to complement the resultsobtained from optical analysis.

An example of using optical analysis for the purpose of identifying theparticles is shown in FIG. 6. Plasma lysed and untreated Bacillusglobigii (BG) spores show different spectra for 10,000 spores. Theplasma lysed spectra shows peaks at 475 cm-1 and 1100 cm-1, while thecontrol spore group does not have peaks in these areas. The 1100 cm-1peak is believed to be the inner carbohydrate layers exposed by theionized gas lysis which peels away the spore coat. The outer spore coatsare comprised of proteinaceous materials. The source of the 475 cm-1peak is unknown, but the peak was also shown by previous NationalInstitute of Standards and Technology (NIST) spectra. In summary, the IRspectrum for BG spores is unique and by virtue of their carbohydratecontent show strong absorption around 1100 cm-1. BG spores have thesebiomarkers but other biological material and interferents do not.Therefore, plasma lysis coupled with IR spectroscopy should provide arapid, definitive detection approach.

The apparatus of the first embodiment allows for redundant confirmationof single particles, which reduces or eliminates false positives. Thelevels of redundant confirmation may include:

First Detection—Sample interrogation via the optical particle analyzerof the particle characterizes the particle by size and threat potential(i.e. surface composition).

Second Detection—Electrosprayed immunoassays that attach totarget-binding sites of particles facilitate optical detection ofnucleic acids or other molecular targets such as proteins.

Third Detection—IR spectroscopic interrogation of the charged particleanalyzer is enhanced by sample enrichment and the reduction inbackground clutter.

Fourth Detection—Plasma lysis (i.e. ionized gas treatment) of theparticles further enhances spectroscopic interrogation as well asextraction of nucleic acids and proteins for subsequent identification,and is especially useful when dealing with disguised particles.

Fifth Detection—The plasma lysis processing makes nucleic acids,proteins, and other biomarkers available for extraction into fluid. Theextractable biomaterials such as the nucleic acids, proteins, and otherbiomarkers may be characterized using polymerase chain reaction (PCR),antigen assay, and other biosensor techniques.

The present invention comprises a method of sorting and analyzingparticles in an aerosol. The method generally comprises collecting anambient aerosol; removing a portion of the fluid from the sample so asto produce a concentrated aerosol; exposing the concentrated aerosol toa laser, wherein the laser collides with the particles and scattersand/or absorbs; measuring the scattering/absorption of the laser;spraying the selected particles with an electrospray fluid; separatingthe electrosprayed particles from the non-electrosprayed particles; andcollecting the electrosprayed particles and analyzing the electrosprayedparticles.

The method will be described in further detail below, but it is notedthat descriptions of some features of the method that overlap with theapparatus described above may not be repeated.

In the first step of the method, a sample of ambient aerosol iscollected. By collected it is meant any means in which a sample of theambient aerosol may be drawn into the apparatus to be subjected to thesubsequent steps of the method independently of the ambient aerosol.

In the second step of the method, the collected aerosol is concentratedby removing a portion of the fluid from the aerosol, preferably withoutalso removing any of the particles contained in the aerosol. By reducingthe amount of fluid in the aerosol, the aerosol becomes moreconcentrated with particles and subsequent treatment of the particles iseasier to perform. In a preferred embodiment, more than about 50% of thefluid in the ambient aerosol sample is removed and more preferably morethan about 90% of the fluid is removed.

The step of removing fluid from the aerosol sample may also be combinedwith removing large particles from the aerosol sample. The targetedparticles for subsequent treatment in the method are normally of a smallsize, and therefore the first step towards separating and collectingselected small particles may be achieved by removing both excess fluidand particles that are larger than a certain size. Specialized design ofa virtual impactor aerosol concentrator may achieve very sharp cut-offpoints for the particles based on their size and density.

After a concentrated aerosol has been produced, the next step in themethod is to expose the concentrated aerosol to a laser. The laser lightcollides and/or absorbs with the particles in the aerosol, thus causingthe laser light to scatter or absorb to varying degrees based upon thecharacteristics of the particle. These measurements are used to analyzeand perform a first identification of the particle.

In a preferred embodiment, a single laser is split into two beams, andthe pulsed or continuous laser beams scatter and/or absorb aftercolliding with the particles. By measuring the scattering and/orabsorption, as well as the velocity, size and particle fluorescentsignature of the particles, a first identification may be achieved. Theparticles may optionally be pretreated to enhance the scattering byadding amino acid binding material to enhance fluorescence.

The measurements of the first identification and corresponding datainterpreted therefrom may be used in the step of electrospraying atleast one particle of interest with at least one electrospray fluid. Theelectrospray fluid preferably coats the particle and adds a charge tothe particle. The electrospray fluid may be any suitable material foradding a charge to a particle and also may include an immunoassay ornanoassay that attaches to the particle as discussed above.

The measurements taken from the first identification may be used todetermine when and at which particles to spray the electrospray fluid.In a preferred embodiment, the velocity measurement of the particle ofinterest is used to time the firing of the electrospray nozzle by alsoknowing the distance between where the particle collides with the laserand where the electrospray fluid is fired. The particle diametermeasurement may be used to determine at which particles to fire theelectrospray fluid. For example, the method can be adjusted such thatonly particles with a predetermined size are coated with an electrosprayfluid. In this manner, any particles not having the specified size arenot electrosprayed, while the particles with the designated size areelectrosprayed using the velocity measurement discussed previously. Thefluorescent signature of the particles determined from the firstidentification may be used for identification purposes and may thereforebe used to determine at which particles to fire an electrospray fluid.Additionally, it should be noted that, where multiple electrosprayfluids may be fired, the size and fluorescent signature measurement maybe used to determine which electrospray fluid to fire at the particle.

In order to assure that the electrospray fluid coats only the selectedparticle, the method of the present invention preferably requires thatthe electrospraying be initiated and terminated in less than about onemillisecond. By requiring a short burst of electrospray fluid, thechance of coating a particle not intended to be coated is significantlydecreased. Thus, the efficiency of the sorting is improved.

In the next step of the method, the electrosprayed particles areseparated from the non-electrosprayed particles and collected. In apreferred embodiment, the separation of electrosprayed particles fromnon-electrosprayed particles is achieved due to the charge that has beenadded to the electrosprayed particles. In a preferred embodiment, afirst charged particle analyzer is provided that will attract chargedparticles due to electrostatic forces, and thereby sort theelectrosprayed particles from the non-electrosprayed particles. Theelectrosprayed particles are attracted towards and settle on a firstcharged particle analyzer, while the non-electrosprayed particlescollect in a separate area.

In the next step of the method, the electrosprayed particles which havebeen separated from non-electrosprayed particles and which have beencollected on a first charged particle analyzer undergo a second,redundant analysis. The aim of the second analysis is to confirm theidentity of the particles of interest, and any suitable means foranalyzing the particles may be used. In the case of a disguisedparticle, the present method comprises removing the disguise by plasmalysis of the coating of the disguised particles. The plasma lysisinvolves subjecting the disguised particles to an ionizing gas thatbreaches the coating of the disguised particle, thereby exposing thepayload or interstitial contents which may be chromosomal nucleic acidand proteins in the case of biological particles.

Once these components of the payload have been exposed, the method asdescribed above may be repeated. For example, optical analysis such asIR spectroscopy may be performed on the exposed particles, followed byelectrospraying, sorting and a second redundant analysis on a chargedparticle analyzer. The IR spectrum obtained during optical analysis maybe used to determine biomarkers unique to the particle, such as thecarbohydrates in the BG spores discussed above. In addition, the payloadmay be further identified by extracting chromosomal nucleic acid exposedby the plasma lysis and performing polymerase chain reaction (PCR)replication.

The foregoing description of the invention has been presented describingcertain operable and preferred embodiments. It is not intended that theinvention should be so limited since variations and modificationsthereof will be within the skill of those skilled in the art, all ofwhich are within the spirit and scope of the invention.

1. An apparatus for the redundant analysis of particles in an aerosol,comprising: a first aerosol concentrator; a first optical analyzerdownstream from and attached to said first aerosol concentrator; a firstelectrosprayer downstream from and attached to said first opticalanalyzer; and a first charged particle analyzer downstream from andattached to said first electrosprayer.
 2. The apparatus as claimed inclaim 1, wherein said first aerosol concentrator comprises a firstradial axisymmetrical virtual impactor.
 3. The apparatus as claimed inclaim 1, wherein said first optical analyzer comprises a first laserselected from the group consisting of infrared lasers, ultravioletlasers, and combinations thereof, and said first laser is adapted toproduce a first laser beam.
 4. The apparatus as claimed in claim 3,further comprising a beam splitter that is adapted to split said firstlaser beam.
 5. The apparatus as claimed in claim 3, wherein said firstoptical analyzer further comprises a first detector that is adapted tomeasure scattering and absorption of said first laser beam.
 6. Theapparatus as claimed in claim 5, wherein said first optical analyzer isadapted to make a first analysis of said measured scattering andadsorption, and said first optical analyzer is further adapted totrigger said first electrosprayer to spray a first electrospray fluid ona first particle of interest.
 7. The apparatus as claimed in claim 6,wherein said first charged particle analyzer is adapted attract saidfirst particle of interest, and to perform a second analysis of saidfirst particle of interest.
 8. The apparatus as claimed in claim 6,wherein said first charged particle analyzer is adapted attract saidfirst particle of interest, and to perform a plasma lysis of said firstparticle of interest, thereby exposing a first payload of said firstparticle of interest.
 9. The apparatus as claimed in claim 8, furthercomprising: a second optical analyzer that is adapted to receive saidfirst payload; a second electrosprayer downstream from and attached tosaid second optical analyzer; and a second charged particle analyzerdownstream from and attached to said second electrosprayer.
 10. Theapparatus as claimed in claim 3, wherein said first aerosol concentratorcomprises a first radial axisymmetrical virtual impactor and a secondradial axisymmetrical virtual impactor, said first optical analyzerfurther comprises a second laser selected from the group consisting ofinfrared lasers, ultraviolet lasers, and combinations thereof, and saidsecond laser is adapted to produce a second laser beam, said firstoptical analyzer further comprises a first detector that is adapted tomeasure scattering and absorption of said first laser beam and saidsecond laser beam, and said apparatus is further adapted to perform abiosensor technique on said first particle of interest, wherein saidbiosensor technique is selected from the group consisting of polymerasechain reaction (PCR), antigen assay, and combinations thereof.
 11. Amethod of performing redundant analysis of particles in an aerosolcomprising the steps of: (1) collecting an aerosol comprising particlesand fluid; (2) removing a portion of the fluid from the aerosol so as toproduce a concentrated aerosol; (3) exposing the particles in theconcentrated aerosol to a first laser beam; (4) measuring the scatteringand absorption of said first laser beam; (5) selecting a first particleof interest based on a first analysis of said measured scattering andabsorption; (6) spraying said first particle of interest with a firstelectrospray fluid to produce an electrosprayed first particle ofinterest; (7) isolating said electrosprayed first particle of interest;and (8) performing a second analysis of said electrosprayed firstparticle of interest.
 12. The method as claimed in claim 11, whereinsaid concentrated aerosol comprises virtually no particles larger than apredetermined size.
 13. The method as claimed in claim 11, wherein saidfirst analysis comprises the comparison of said measured scattering andabsorption to a previously measured scattering and absorption of apreviously known particle of interest.
 14. The method as claimed inclaim 11, wherein said spraying with said first electrospray fluid isinitiated and terminated in less than about 1 millisecond.
 15. Themethod as claimed in claim 11, wherein there is an electric charge onsaid electrosprayed first particle of interest.
 16. The method asclaimed in claim 11, wherein said second analysis comprises theperformance of a biosensor technique on said first particle of interest,wherein said biosensor technique is selected from the group consistingof polymerase chain reaction (PCR), antigen assay, and combinationsthereof.
 17. The method as claimed in claim 11, wherein said exposingfurther comprises the exposure of said particles in said concentratedaerosol to a second laser beam, and said spraying further comprises asecond electrospray fluid.
 18. The method as claimed in claim 11,wherein said second analysis comprises performing a plasma lysis of saidelectrosprayed first particle of interest, thereby exposing a firstpayload of said electrosprayed first particle of interest.
 19. Themethod as claimed in claim 18, further comprising the steps of: (9)exposing said first payload to a second laser beam; (10) measuring thescattering and absorption of said second laser beam; (11) selecting asecond particle of interest based on a third analysis of said measuredscattering and absorption of said second laser beam; (12) spraying saidsecond particle of interest with a second electrospray fluid to producean electrosprayed second particle of interest; (13) isolating saidelectrosprayed second particle of interest; and (14) performing a fourthanalysis of said electrosprayed second particle of interest.
 20. Themethod as claimed in claim 19, wherein said fourth analysis comprisesthe performance of a biosensor technique on said electrosprayed secondparticle of interest, wherein said biosensor technique is selected fromthe group consisting of polymerase chain reaction (PCR), antigen assay,and combinations thereof.